Trichomonas vaginalis Lipophosphoglycan Exploits Binding to Galectin-1 and -3 to Modulate Epithelial Immunity [Molecular Bases of Disease]

November 20th, 2015 by

Trichomoniasis is the most common non-viral sexually transmitted infection caused by the vaginotropic extracellular protozoan parasite Trichomonas vaginalis. The infection is recurrent, with no lasting immunity, often asymptomatic and linked to pregnancy complications and risk of viral infection. The molecular mechanisms of immune evasion by the parasite are poorly understood. We demonstrate that galectin-1 and -3 are expressed by the human cervical and vaginal epithelial cells and act as pathogen-recognition receptors for the ceramide phospho-inositol glycan core (CPI-GC) of the dominant surface protozoan lipophosphoglycan (LPG). We used an in-vitro model with siRNA galectin knockdown epithelial clones, recombinant galectins, clinical trichomonas isolates and mutant protozoan derivatives to dissect the function of galectin-1 and -3 in the context of trichomonas infection. Galectin-1 suppressed chemokines that facilitate recruitment of phagocytes, which can eliminate extracellular protozoa (IL-8) or bridge innate to adaptive immunity (MIP-3α and RANTES). Silencing galectin-1 increased and adding exogenous galectin-1 suppressed chemokine responses to trichomonas or CPI-GC/LPG. In contrast, silencing galectin-3 reduced IL-8 response to LPG. Live trichomonas depleted the extracellular levels of galectin-3. Clinical isolates and mutant Trichomonas CPI-GC that had reduced affinity to galectin-3 but maintained affinity to galectin-1 suppressed chemokine expression. Thus via CPI-GC binding trichomonas is capable of regulating galectin bioavailability and function to the benefit of its parasitic survival. These findings suggest novel approaches to control trichomoniasis and warrant further studies of galecitin-binding diversity among clinical isolates as a possible source for symptom disparity in parasitic infections.

Arabidopsis Rab Geranylgeranyltransferases Demonstrate Redundancy and Broad Substrate Specificity in vitro [Plant Biology]

November 20th, 2015 by Shi, W., Zeng, Q., Kunkel, B. N., Running, M. P.

Posttranslational lipid modifications mediate the membrane attachment of Rab GTPases, facilitating their function in regulating intracellular vesicular trafficking. In Arabidopsis, most Rab GTPases have two C-terminal cysteines and potentially can be double geranylgeranylated by heterodimeric Rab geranylgeranyltransferases (Rab-GGTs). Genes encoding two putative α subunits and two putative β subunits of Rab-GGTs have been annotated in the Arabidopsis thaliana genome, but little is known about Rab-GGT activity in Arabidopsis. In this study, we demonstrate that four different heterodimers can be formed between putative Arabidopsis Rab-GGT α subunits RGTA1/RGTA2 and β subunits RGTB1/RGTB2, but only RGTA1-RGTB1 and RGTA1-RGTB2 exhibit bona fide Rab-GGT activity, and they are biochemically redundant in vitro. We hypothesize that RGTA2 function might be disrupted by a 12-amino acid insertion in a conserved motif. We present evidence that Arabidopsis Rab-GGTs may have preference for prenylation of C-terminal cysteines in particular positions. We also demonstrate that Arabidopsis Rab-GGTs can prenylate not only a great variety of Rab GTPases in the presence of Rab escort protein (REP), but, unlike Rab-GGT in yeast and mammals, can also certain non-Rab GTPases independent of REP. Our findings may help explain some of the phenotypes of Arabidopsis protein prenyltransferase mutants.

Advanced glycation end products (AGE) potently induce autophagy through activation of RAF kinase and NF-KAPPA B [Molecular Bases of Disease]

November 19th, 2015 by Verma, N., Manna, S. K.

Advanced glycation end products (AGE) accumulate in diabetic patients and aging people due to high amounts of 3- or 4-carbon sugars derived from glucose and thereby causing multiple consequences including inflammation, apoptosis, obesity and age-related disorders. It is important to understand the mechanism of AGE-mediated signaling leading to activation of autophagy (self-eating) that might result in obesity. We have detected AGE as one of the potent inducers of autophagy compared to doxorubicin and TNF. AGE-mediated autophagy is inhibited by suppression of PI3 kinase and potentiated by autophagosome maturation blocker, bafilomycin. It increases autophagy in different cell types and that correlates with the expression of it receptor, RAGE. LC3B, the marker for autophagosome is shown to increase upon AGE stimulation. AGE-mediated autophagy is suppressed partially by inhibitor of NF-κB, PKC, or ERK alone and significantly in combination. AGE increases SREBP activity that leads to increase in lipogenesis. Though AGE-mediated lipogenesis is affected by autophagy inhibitor, AGE-mediated autophagy is not influenced by lipogenesis inhibitor, suggesting that the turnover of lipid droplets overcomes the autophagic clearance. For the first time, we are providing data that AGE induces several cell signaling cascades, like NF-κB, PKC, ERK, and MAPK, which are involved in autophagy and simultaneously help in accumulating lipid droplets which are not effectively cleared by autophagy, thus follows obesity.

The Relay-Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II [Cell Biology]

November 19th, 2015 by Bloemink, M. J., Melkani, G. C., Bernstein, S. I., Geeves, M. A.

The interface between relay and converter domain of muscle myosin is critical for optimal myosin performance. Using Drosophila melanogaster indirect flight muscle S1 we performed a kinetic analysis of the effect of mutations in the converter and relay domain. Introduction of a mutation (R759E) in the converter domain inhibits the steady-state ATPase of myosin S1, whereas an additional mutation in the relay domain (N509K) is able to restore the ATPase towards wild-type values. The S1- R759E construct showed little effect on most steps of the actomyosin ATPase cycle. The exception was a 25-30% reduction in the rate constant of the hydrolysis step, the step coupled to the cross-bridge recovery stroke and involving a change in conformation at the relay/converter domain interface. Significantly the double mutant restored the hydrolysis step to values similar to the wild-type myosin. Modelling the relay/converter interface suggests a possible interaction between converter residue 759 and relay residue 509 in the actin-detached conformation, which is lost in R759E but is restored in N509K/R759E. This detailed kinetic analysis of Drosophila myosin carrying the R759E mutation shows that the interface between the relay loop and converter domain is important for fine-tuning myosin kinetics, in particular ATP-binding and hydrolysis.

Increased Glucose-induced Secretion of Glucagon-like Peptide-1 in Mice Lacking the Carcino-Embryonic Antigen-related Cell Adhesion Molecule 2 [Metabolism]

November 19th, 2015 by

Carcinoembryonic antigen-related cell adhesion molecule 2 (CEACAM2) regulates food intake as demonstrated by hyperphagia in mice with Ceacam2 null mutation (Cc2-/-). The current studies investigated whether CEACAM2 also regulates insulin secretion. Ceacam2 deletion caused an increase in beta cell secretory function, as assessed by hyperglycemic clamp analysis, without affecting insulin response. Although CEACAM2 is expressed in pancreatic islets predominantly in non-beta cells, basal plasma levels of insulin, glucagon and somatostatin, islet areas, and glucose-induced insulin secretion in pooled Cc2-/- islets were all normal. Consistent with immunofluorescence analysis showing CEACAM2 expression in distal intestinal villi, Cc2-/- mice exhibited a higher release of oral glucose-mediated GLP-1, an incretin that potentiates insulin secretion in response to glucose. Compared to wild type, Cc2-/- mice also showed a higher insulin excursion during oral glucose tolerance test. Pretreating with exendin (9-39), a GLP-1 receptor antagonist, suppressed the effect of Ceacam2 deletion on glucose-induced insulin secretion. Moreover, GLP-1 release into the medium of GLUTag entero-endocrine cells was increased with siRNA-mediated Ceacam2 downregulation in parallel to increase in Ca2+ entry through L-type voltage-dependent Ca2+ channels. Thus, CEACAM2 regulates insulin secretion, at least in part, by a GLP-1 mediated mechanism, independent of confounding metabolic factors.
  • Posted in Journal of Biological Chemistry, Publications
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Mitochondrial Ribosomal Protein L12 is required for POLRMT Stability and Exists as Two Forms Generated by Alternative Proteolysis During Import [Cell Biology]

November 19th, 2015 by

To translate the thirteen mtDNA-encoded mRNAs involved in oxidative phosphorylation (OXPHOS), mammalian mitochondria contain a dedicated set of ribosomes comprising rRNAs encoded by the mitochondrial genome and mitochondrial ribosomal proteins (MRPs) that are encoded by nuclear genes and imported into the matrix. In addition to their role in the ribosome, several MRPs have auxiliary functions or have been implicated in other cellular processes like cell cycle regulation and apoptosis. For example, we have shown that human MRPL12 binds and activates mitochondrial RNA polymerase (POLRMT), and hence has distinct functions in the ribosome and mtDNA transcription. Here we provide concrete evidence that there are two mature forms of mammalian MRPL12 that are generated by a two-step cleavage during import, involving efficient cleavage by mitochondrial processing protease (MPP) and a second inefficient or regulated cleavage by mitochondrial intermediary protease (MIP). We also show that knock-down of MRPL12 by RNAi results in instability of POLRMT, but not other primary mitochondrial transcription components, and a corresponding decrease in mitochondrial transcription rates. Knock-down of MRPL10, the binding partner of MRPL12 in the ribosome, results in selective degradation of the mature long form of MRPL12, but has no effect on POLRMT. We propose that the two forms of MRPL12 are involved in homeostatic regulation of mitochondrial transcription and ribosome biogenesis that likely contribute to cell cycle, growth regulation and longevity pathways to which MRPL12 has been linked.
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Peptide Bond Synthesis by a Mechanism Involving an Enzymatic Reaction and a Subsequent Chemical Reaction [Metabolism]

November 19th, 2015 by

We recently reported that an amide bond is unexpectedly formed by an acyl-CoA synthetase (which catalyzes the formation of a carbon-sulfur bond) when a suitable acid and L-cysteine are used as substrates. DltA, which is homologous to the adenylation domain of nonribosomal peptide synthetase, belongs to the same superfamily of adenylate-forming enzymes, which comprises many kinds of enzymes including the acyl-CoA synthetases. Here, we demonstrate that DltA synthesizes not only N-(D-alanyl)-L-cysteine (a dipeptide) but also various oligopeptides. We propose that this enzyme catalyzes peptide synthesis by the following unprecedented mechanism: (i) the formation of S-acyl-L-cysteine as an intermediate via its "enzymatic activity" and (ii) subsequent "chemical" S→N acyl transfer in the intermediate, resulting in peptide formation. Step (ii) is identical to the corresponding reaction in native chemical ligation (NCL), a method of chemical peptide synthesis, whereas step (i) is not. To the best of our knowledge, our discovery of this peptide synthesis mechanism involving an enzymatic reaction and a subsequent chemical reaction is the first such one to be reported. This new process yields peptides without the use of a thioesterified fragment, which is required in NCL. Together with these findings, the same mechanism-dependent formation of N-acyl-compounds by other members of the above-mentioned superfamily demonstrated that all members most likely form peptide/amide compounds by using this novel mechanism. Each member enzyme acts on a specific substrate, thus, not only the corresponding peptides but also new types of amide compounds can be formed.

Leishmania donovani encodes a functional selenocysteinyl-tRNA synthase [Gene Regulation]

November 19th, 2015 by Manhas, R., Gowri, V. S., Madhubala, R.

The synthesis of selenocysteine, the 21st amino acid occurs on its transfer RNA (tRNA), tRNASec. tRNASec is initially aminoacylated with serine by seryl-tRNA synthetase and the resulting seryl moiety is converted to phosphoserine by O-phosphoseryl-tRNA kinase (PSTK) in eukaryotes. The selenium donor, selenophosphate is synthesized from selenide and ATP by selenophosphate synthetase. Selenocysteinyl-tRNA synthase (SepSecS) then uses the O-phosphoseryl-tRNASec and selenophosphate, to form Sec-tRNASec in eukaryotes. Here, we report the characterization of selenocysteinyl-tRNA synthase from L. donovani. Kinetoplastid SepSecS enzymes are phylogenetically closer to worm SepSecS. LdSepSecS was found to exist as a tetramer. Leishmania SepSecS enzyme was found to be active and able to complement the ∆selA deletion in E. coli JS1 strain only in the presence of archaeal PSTK, indicating the conserved nature of the PSTK-SepSecS pathway. LdSepSecS was found to localize in the cytoplasm of the parasite. Gene deletion studies indicate that Leishmania SepSecS is dispensable for the parasite survival. The parasite was found to encode three selenoproteins; which expressed only in the presence of SepSecS. Selenoproteins of L. donovani are not required for the growth of the promastigotes. Auranofin, a known inhibitor of selenoprotein synthesis showed same sensitivity towards the wild-type and the null mutants suggesting its effect is not through binding to selenoproteins. The 3-D structural comparison indicates that the human and Leishmania homologs are structurally highly similar but their association modes leading to tetramerization seem different

Cooperative interactions of oligosaccharide and peptide moieties of a glycopeptide derived from IgE with galectin-9 [Immunology]

November 18th, 2015 by

We previously showed that galectin-9 suppresses degranulation of mast cells through protein-glycan interaction with IgE. To elucidate the mechanism of the interaction in detail, we focused on identification and structural analysis of IgE glycans responsible for the galectin-9-induced suppression using mouse monoclonal IgE (TIB-141). TIB-141 in combination with the antigen induced degranulation of RBL-2H3 cells, which was almost completely inhibited by human and mouse galectin-9. Sequential digestion of TIB-141 with lysyl endopeptidase and trypsin resulted in the identification of a glycopeptide (H-Lys13-Try3; 48 amino acid residues) with a single N-linked oligosaccharide near the N-terminus capable of neutralizing the effect of galectin-9 and another glycopeptide with two N-linked oligosaccharides (H-Lys13-Try1; 16 amino acid residues) having lower activity. Enzymatic elimination of the oligosaccharide chain from H-Lys13-Try3 and H-Lys13-Try1 completely abolished the activity. Removal of the carboxy-terminal 38 amino acid residues of H-Lys13-Try3 with glutamyl endopeptidase, however, also resulted in loss of the activity. We determined the structures of N-linked oligosaccharides of H-Lys13-Try1. The galectin-9-binding fraction of oligosaccharides released with peptide-N-glycosidase F contained asialo- and mono-sialylated bi/tri-antennary complex-type oligosaccharides with a core fucose residue. The structures of the oligosaccharides were consistent with the sugar-binding specificity of galectin-9, while the nonbinding fraction contained monosialylated and disialylated biantennary complex-type oligosaccharides with a core fucose residue. Although the oligosaccharides linked to H-Lys13-Try3 could not be fully characterized, these results indicate the possibility that cooperative binding of oligosaccharide and neighboring polypeptide structures of TIB-141 to galectin-9 affects the overall affinity and specificity of the IgE-lectin interaction.

The A-kinase anchoring protein GSKIP regulates GSK3{beta} activity and controls palatal shelf fusion in mice [Developmental Biology]

November 18th, 2015 by

A-kinase anchoring proteins (AKAPs) represent a family of structurally diverse proteins, all of which bind protein kinase A (PKA). A member of this family is Glycogen synthase kinase 3β (GSK3β) interaction protein (GSKIP). GSKIP interacts with PKA and also directly with GSK3β. The physiological function of the GSKIP protein in vivo is unknown. We developed and characterized a conditional knockout mouse model and found that GSKIP deficiency caused lethality at birth. Embryos obtained through Caesarean section at embryonic day E18.5 were cyanotic, suffered from respiratory distress, and failed to initiate breathing properly. Additionally, all GSKIP-deficient embryos showed an incomplete closure of the palatal shelves accompanied by a delay in ossification along the fusion area of secondary palatal bones. On the molecular level, GSKIP deficiency resulted in decreased phosphorylation of GSK3β at Ser9 starting early in development (E 10.5), leading to enhanced GSK3β activity. At embryonic day 18.5 GSK3β activity decreased to levels close to that of wild type. Our findings reveal a novel, crucial role for GSKIP in the coordination of GSK3β signaling in palatal shelf fusion.